Development of Molecular Marker Linked to Seed Hardness in Pomegranate Using Bulked Segregant Analysis

The pomegranate (Punica granatum L.) is one of the fruit species with the oldest cultural history. There are many traits to determine the quality of pomegranate fruits. Among them, soft-seeded feature of pomegranate fruit is important trait for the market value of the fruit. For this reason, the demand for pomegranate varieties with soft seeds has been increasing, especially in recent years. In this study, molecular markers associated with seed hardness were developed to distinguish pomegranate cultivars with soft-seeded feature based on genomic DNA at the early stages of the pomegranate breeding process. For this purpose, pomegranate genotypes and/or cultivars from the population involved in reciprocal crosses of hard-seeded Ernar, medium-hard-seeded Hicaznar, and soft-seeded Fellahyemez cultivars were grouped as soft-seeded or hard-seeded. Further, leaf samples were collected from individuals belonging to each group. Then, the genomic DNA was isolated from each plant separately, and equal amount of genomic DNA from individuals with the similar seed hardness were mixed for bulked segregant analysis (BSA). The bulked genomic DNAs of opposite characters were analyzed by polymerase chain reaction (PCR) using random decamer primers to develop random amplified polymorphic DNA (RAPD) markers associated with soft-seeded or hard-seeded pomegranates. A total of three RAPD markers were determined to distinguish the individuals having soft- or hard-seeded pomegranate genotypes and/or cultivars. As a result of the comparison of the DNA sequences of these RAPD markers, insertion-deletions (inDels) primers were designed to developed and validate a PCR assay to distinguish the soft- and hard-seeded pomegranate genotypes/cultivars from each other. The molecular markers developed in this study will enable us to distinguish soft-seeded pomegranate types easily in a short time at the early stages of the pomegranate breeding programs.


Introduction
Pomegranate, one of the ancient fruit species, has been consumed both fresh and juiced for thousands of years. The demand for the production and consumption of pomegranate fruit has been increased in recent years due to its important bioactive properties such as antimicrobial [1], antiparasitic [2], antiviral [3], and anticancer [4] activities. One of the most important reasons for the increase in its production is that it is not selective in terms of climate and soil. In addition, its rich nutritional value, positive effects on human health, and the long storage period and releasing time to the market when the other fruits are not abundant can be counted among the other reasons for the increases in production and consumption of pomegranate.
The edible part of the pomegranate fruit is called aril and constitutes 45-52% of the total fruit weight. Aril consists of a white, yellow, pink, and red fleshy, juicy part and the seeds inside consist of the embryo with a shell surrounding it [5][6][7]. It has a sour or sweet Life 2023, 13, 1123 3 of 12 (MAS). Determination of the characteristics of the fruits could be possible without waiting for an average of 5-10 years for bearing fruits in perennial plants using MAS. Due to this time-saving process, it is also possible to reduce the space, cost, and labor [25,26]. In order to use all these advantages of MAS in pomegranate breeding as well, it is necessary to develop molecular markers associated with economically important traits in pomegranate. Therefore, in this study, molecular markers associated with the soft-and hard-seed traits in pomegranates were developed. Different molecular markers have been developed to date using different techniques, and each has different advantages. Among them, random amplified polymorphic DNA (RAPD) marker has been used for many years in many agricultural products because it can be used without the need for sequence information and requires very small amount of DNA. In this study, the RAPD technique combined with bulked segregant analysis (BSA) was used to find the marker(s) associated with soft-and hard-seed characteristics in pomegranates.

Plant Material
Pomegranate genotypes were obtained from hybridization of different combinations of hard-seeded Ernar, medium-hard-seeded Hicaznar, and soft-seeded Fellahyemez cultivars in 1990 in Turkey. These crosses were made in order to develop new pomegranate varieties and phenological, morphological, and pomological characteristics of the population were determined by different researchers at different time periods [27,28] at the Western Mediterranean Agricultural Research Institute in Turkey. Among this population, 94 pomegranate genotypes and their parents were used as plant material in this study. Genotypes previously scored as hard-seeded or soft-seeded were re-scored by different experts in two harvest seasons in this study according to Yazici and Sahin [28]. Based on these evaluations, new phenotypical groups were formed and only hard-seeded and soft-seeded genotypes were used as plant material in this study.

Polymorphisms Analysis by RAPD and SSR Primers
The bulked DNA samples containing genomic DNAs of pomegranate genotypes with hard-and soft-seeded were analyzed by polymerase chain reaction (PCR) method using 260 random primers (Operon Technologies Inc., Alameda, CA, USA) and 24 different SSR primer pairs previously developed for pomegranate [13,[30][31][32][33][34][35][36][37][38][39]. RAPD primers were primarily selected based on previous studies in pomegranate and random primers known to give polymorphism in pomegranate fruit were used first, and then random selections were made among other Operon random primers. A list of all primers used in this study is given in Supplementary Files Tables S1 and S2. In each random primer or SSR primer pair, a PCR mix was prepared with 1X PCR buffer solution (50 mM KCl at 25 • C, 10 mM Tris HCl pH 9.0, 1% Triton X-100) 2.5 mM MgCl 2 , 0.2 mM dNTP, 10 µM RAPD primer or SSR primers, 1.25 U Taq polymerase (Thermo Fisher, USA), and 25-75 ng DNA. The PCR amplification was performed using MJ Mini PTC1148 and ICycler (Bio-Rad, USA) thermocyclers. PCR reactions were performed in thin-walled PCR plates or tubes in two different thermocyclers programmed as an initial denaturation of 5 min at 94 • C, followed Life 2023, 13, 1123 4 of 12 by 35-40 cycles of denaturation for 30 sec-1 min at 94 • C, primer annealing for 30 sec-1 min at 35-65 • C, and primer extension for 2-3 min at 72 • C followed by one cycle of at 72 • C for 10 min for final primer extension. After the amplification, RAPD-PCR products were separated on a 1.5-3% agarose gel in 1 × TAE buffer [50 × (2 M Tris-base, 0.1 M EDTA pH 8.0, 5.7% glacial acetic acid)] and photographed under UV light using Mini BIS-Pro (DNR, Neve Yamin, Israel) gel imaging system. On the other hand, PCR products of SSR were separated using Qsep 100 fragment analyzer system (BiOptic, New Taipei City, Taiwan).

Scoring and Marker Analysis
Only the intense and clear bands consistently produced using genomic DNAs isolated at different time and different PCR machines were considered to be polymorphic and selected for further analysis. To confirm the consistency of a selected polymorphic bands, repetitive PCR reactions were performed using genomic DNAs of individuals constituting the bulked groups. Validation of polymorphic bands was performed using DNAs from the other members of progeny population, but not used in bulk analysis.

Conversion of RAPD Markers to Insertion-Deletions (InDels) Markers
Polymorphic DNA fragments associated with hard and soft seed traits were cut from agarose gel and purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The purified DNA fragments were cloned into the pGEM-T easy vector using the T-A cloning method with pGEM-T easy PCR cloning system (Promega, Madison, WI, USA), as suggested by the manufacturer's instructions to determine their sequences.
The clones were transferred to Escherichia coli JM109 cells by heat shock transformation and screening of the resulting bacterial colonies was performed by colony PCR using M13 forward (F) and reverse (R) primers located outside of the T-A cloning site. Colonies showing positive result by colony PCR were grown in liquid LB medium with ampicillin, and purification of the plasmid DNA was performed using the Mini Prep Plasmid DNA isolation Kit (Qiagen, Germany), according to the manufacturer's instructions. For the confirmation of the presence of additional DNA in the plasmid, the purified plasmids were digested using the EcoRI enzyme (New England Biolab, Ipswich, MA, USA). Sequencing of plasmids containing polymorphic DNA fragments was carried out by bidirectional Sanger sequencing using universal primers, M13 F, and M13 R. Sequences of the obtained polymorphic DNA fragments were analyzed using the Vector NTI suite program (Infor-Max, Frederick, MD, USA). BLASTN and BLASTX applications of the National Center for Biotechnology Information (NCBI) were used to compare the sequences with the other sequences in GenBank at the nucleotide and protein level, respectively. Comparisons of the sequences with each other were performed using the BLAST-alignment module of the NCBI program. Based on the sequence comparisons, the InDel primers (forward 5 GGCCCTCACATATTAAGTTCAC 3 and reverse 5 GTATCTTGAAAGTCAATGAGCC 3 ) were designed. These InDel primers were used with 10 µM concentration in a 25-µL PCR reaction mixture as described in Section 2.3. The amplification of these PCR was performed in MJ Mini PTC1148 thermocycler (Bio-Rad, Hercules, CA, USA) with conditions at 94 • C for 5 min for initial denaturation for one cycle, followed by 39 cycles of 94 • C for 30 s denaturation, 55 • C for 30 s primer annealing, 72 • C for 30 s primer extension, and a final primer extension at 72 • C for 10 min. PCR products were separated and visualized as described in Section 2.3 by preparing a 3% agarose gel.

Results
Phenotypical groupings of the selected population based on their seed hardness were re-evaluated for two years in row in this study, and new groups were formed. Bulked DNA samples representing opposite group of individuals', hard-seeded, and soft-seeded were formed based on this new grouping. As a result of PCR analyzes using bulked DNA samples and SSR primer pairs, no polymorphic band or marker distinguishing hard-and soft-seeded individuals was determined. However, RAPD-PCR analysis using different Life 2023, 13, 1123 5 of 12 random primers (OPE-19, OPD-17, OPAD-18, OPAY-09, OPAE-14, OPC-12, OPR-15, OPK-12, OPAI-08) produced 10 polymorphic bands indicating the differences between two bulked samples. To test the consistency of the obtained polymorphic bands as well as their ability to distinguish between hard-and soft-seeded individuals, genomic DNAs of individuals analyzed several times by RAPD-PCR using these primers. After repeated RAPD-PCR analyses, polymorphic bands were consistently obtained only from OPK-12 and OPAI-08 primers. Moreover, the validity of these polymorphic bands was then confirmed by PCR analyses of individual genomic DNAs constituting the bulks. After multiple PCR analyses, three polymorphic bands produced by these two different primers that were able to distinguish individuals with hard-and soft-seeded pomegranate genotypes were identified. Two of these polymorphic bands, about 550 and 600 bp, were obtained from RAPD-PCR amplification using the OPK-12 primer. These two polymorphic bands were very close to each other in size, but one band was only in soft-seeded individuals with a size of 550 bp, and the other band was only present in hard-seeded individuals with a size of 600 bp band. These markers are named as RAPDSS1 and RAPDHS1 markers, respectively ( Figure 1). On the other hand, only one polymorphic band was obtained as a result of RAPD-PCR analysis with OPAI-08 primer. The analyses clearly showed that the 700 bp polymorphic band was only present in individuals in the soft-seeded genotype bulks and not in the individuals forming the hard-seeded genotype bulks ( Figure 2). After demonstration of its consistent ability to distinguish soft-and hard-seeded individuals from each other, this polymorphic band was named as RAPDSS2 marker. Validation of all these three molecular markers was performed by PCR reactions using DNAs from the other hard-and soft-seeded individuals which were not included in bulks in the population (Figures 3 and 4). random primers (OPE-19, OPD-17, OPAD-18, OPAY-09, OPAE-14, OPC-12, OP 12, OPAI-08) produced 10 polymorphic bands indicating the differences be bulked samples. To test the consistency of the obtained polymorphic bands as w ability to distinguish between hard-and soft-seeded individuals, genomic DN viduals analyzed several times by RAPD-PCR using these primers. After repea PCR analyses, polymorphic bands were consistently obtained only from O OPAI-08 primers. Moreover, the validity of these polymorphic bands was then by PCR analyses of individual genomic DNAs constituting the bulks. After mu analyses, three polymorphic bands produced by these two different primers able to distinguish individuals with hard-and soft-seeded pomegranate geno identified. Two of these polymorphic bands, about 550 and 600 bp, were obt RAPD-PCR amplification using the OPK-12 primer. These two polymorphic b very close to each other in size, but one band was only in soft-seeded individ size of 550 bp, and the other band was only present in hard-seeded individuals of 600 bp band. These markers are named as RAPDSS1 and RAPDHS1 marke tively ( Figure 1). On the other hand, only one polymorphic band was obtained of RAPD-PCR analysis with OPAI-08 primer. The analyses clearly showed that polymorphic band was only present in individuals in the soft-seeded genotype not in the individuals forming the hard-seeded genotype bulks ( Figure 2). Af stration of its consistent ability to distinguish soft-and hard-seeded individuals other, this polymorphic band was named as RAPDSS2 marker. Validation of all molecular markers was performed by PCR reactions using DNAs from the o and soft-seeded individuals which were not included in bulks in the populatio 3 and 4).  The OPK-12 primer produced polymorphic bands between hard-and soft-seeded individuals. PCR product of 550 bp amplified from a soft-seeded individual (16-99) and 600 bp amplified from a hard-seeded individual (17-21) by OPK-12 primer were excised from the gel and cloned to determine their DNA sequences. After cleaning the vector sequences, 571 and 608 bp clean sequence were obtained from the cloned PCR fragments of genotype 16-99 and 17-21, respectively. The comparison of the nucleotide sequences revealed that both fragments were aligned with each other and there was 33 bp deletion in soft-seeded genotype 16-99 ( Figure 5).     BLASTN analysis showed that 550 bp DNA from the soft-seeded genotype 16-99 matched with uncharacterized cDNA (GenBankacession: XM_031544412.1) of Punica granatum which was submitted the GenBank from sequencing the soft-seeded variety, 'Tunisia'. In addition, the results of BLASTX analysis demonstrated that this protein was found in GenBank as protein number XP_031400272 and its function has not yet been determined. On the other hand, BLAST P analyzes demonstrated that orthologs of this protein has been functioned as 'Calcium-dependent lipid-binding domain-containing protein' in  The OPK-12 primer produced polymorphic bands between hard-and soft-seeded individuals. PCR product of 550 bp amplified from a soft-seeded individual (16-99) and 600 bp amplified from a hard-seeded individual (17-21) by OPK-12 primer were excised from the gel and cloned to determine their DNA sequences. After cleaning the vector sequences, 571 and 608 bp clean sequence were obtained from the cloned PCR fragments of genotype 16-99 and 17-21, respectively. The comparison of the nucleotide sequences revealed that both fragments were aligned with each other and there was 33 bp deletion in soft-seeded genotype 16-99 ( Figure 5). The sequence analysis of the soft-and hard-seeded genotypes revealed 33 bp deletion in the soft-seeded genotype. This region was the source of polymorphisms detected by OPK-12 and the sequences were identical to the coding sequences in the pomegranate genome. Based on sequence information obtained from the cloned RAPD fragments of OPK-12, two primers were designed to differentiate hard-and soft-seeded genotypes to convert the RAPD marker into more reliable sequence based markers in this study ( Figure 5). Since the polymorphisms were based on internal deletion in the soft-seeded genotype, the sequence-characterized marker developed based on these sequences is considered as an insertion/deletion (InDel) marker ( Figure 6).
This InDel marker was first tested by PCR analyzes of the individual genomic DNAs forming the bulks. This PCR analysis clearly showed that while only 156 bp fragment was amplified from hard-seeded genotypes, a 123 bp fragment was amplified from soft-seeded genotypes along with the 156 bp DNA fragment in the population. Later, the ability of this InDel marker to distinguish between hard-and soft-seeded individuals was tested by PCR reactions using DNAs from the other individuals' in the population were not included in the bulk. All these results showed that the primers designed based on sequences obtained from polymorphic RAPD fragments were consistently differentiate soft-and hard-seeded genotypes in the hybrid pomegranate population (Figure 7). BLASTN analysis showed that 550 bp DNA from the soft-seeded genotype 16-99 matched with uncharacterized cDNA (GenBankacession: XM_031544412.1) of Punica granatum which was submitted the GenBank from sequencing the soft-seeded variety, 'Tunisia'. In addition, the results of BLASTX analysis demonstrated that this protein was found in GenBank as protein number XP_031400272 and its function has not yet been determined. On the other hand, BLAST P analyzes demonstrated that orthologs of this protein has been functioned as 'Calcium-dependent lipid-binding domain-containing protein' in perennial trees, Melia azedarach (KAJ4722804.1), and Salix purpurea (KAJ6719349.1). Furthermore, based on the analysis in the Arabidopsis Information Resource (TAIR) database, it was determined that the ortholog of this gene is At1g04540 and its function is defined as 'Calcium-dependent lipid binding (CaLB domain) protein'. Studies in Arabidopsis have also been demonstrated that this gene is expressed during mature pollen stage, germinated pollen stage, flowering stage, petal differentiation and expansion stage, and plant embryo globular stage during plant growth and development.
The sequence analysis of the soft-and hard-seeded genotypes revealed 33 bp deletion in the soft-seeded genotype. This region was the source of polymorphisms detected by OPK-12 and the sequences were identical to the coding sequences in the pomegranate genome. Based on sequence information obtained from the cloned RAPD fragments of OPK-12, two primers were designed to differentiate hard-and soft-seeded genotypes to convert the RAPD marker into more reliable sequence based markers in this study ( Figure  5). Since the polymorphisms were based on internal deletion in the soft-seeded genotype, the sequence-characterized marker developed based on these sequences is considered as an insertion/deletion (InDel) marker ( Figure 6). This InDel marker was first tested by PCR analyzes of the individual genomic DNAs forming the bulks. This PCR analysis clearly showed that while only 156 bp fragment was amplified from hard-seeded genotypes, a 123 bp fragment was amplified from soft-seeded genotypes along with the 156 bp DNA fragment in the population. Later, the ability of this InDel marker to distinguish between hard-and soft-seeded individuals was tested by PCR reactions using DNAs from the other individuals' in the population were not included in the bulk. All these results showed that the primers designed based on sequences obtained from polymorphic RAPD fragments were consistently differentiate soft-and hard-seeded

Discussion
Pomegranate varieties with soft-seeded feature are very few and only found in an area limited to a few ecological regions in the world [9]. For example, it was determined that only 2 of 87 different pomegranate genotypes showed soft seed characteristics [40][41][42]. Similarly, Khadivi and Arab [11] determined that only 17 of the 70 pomegranate genotypes they examined soft-seeded trait. Similar to the results of these studies, only 12 of the 94 genotypes examined in this study showed completely soft-seed characteristics. Further, it has been observed that pomegranates with this soft-seed trait do not show the desired fruit characteristics sufficiently. This result is consistent with the results of other studies, which determined that pomegranate cultivars with soft-seeded features do not have other good fruit quality criteria [9,12]. On the other hand, pomegranates with hardseed features generally have the desired peel and aril color. For example, Hicaznar, a pomegranate variety used in this study and also widely cultivated, has a hard-seeded cultivars although it shows superior characteristics in terms of dark red skin and other fruit characteristics. These findings show that the number of pomegranate cultivars with soft-seeded characteristics is quite low in the world and the most of them do not have other good fruit characteristics. Therefore, it is necessary to develop new pomegranate cultivars with red skin and red aryl color, and also soft-seeded features.
Development of new pomegranate varieties with desired characteristics is very difficult and takes a long period of time by conventional breeding methods. Marker assisted selection (MAS), which is among the new breeding techniques, reduce the amount of time during breeding of new fruit cultivars [43,44]. MAS allows the selection based on the genotype directly without looking at the phenotype of the plants [45]. In order to apply this technique, there is a need to develop molecular markers associated with the desired traits.
Up to date, no molecular marker to be used directly in the breeding process of pomegranates has been identified. However, there are some studies conducted for the identification of molecular markers related with some traits including soft-seed feature. For example, in a study conducted by Sarkhosh et al., [5], a correlation was tried to be

Discussion
Pomegranate varieties with soft-seeded feature are very few and only found in an area limited to a few ecological regions in the world [9]. For example, it was determined that only 2 of 87 different pomegranate genotypes showed soft seed characteristics [40][41][42]. Similarly, Khadivi and Arab [11] determined that only 17 of the 70 pomegranate genotypes they examined soft-seeded trait. Similar to the results of these studies, only 12 of the 94 genotypes examined in this study showed completely soft-seed characteristics. Further, it has been observed that pomegranates with this soft-seed trait do not show the desired fruit characteristics sufficiently. This result is consistent with the results of other studies, which determined that pomegranate cultivars with soft-seeded features do not have other good fruit quality criteria [9,12]. On the other hand, pomegranates with hard-seed features generally have the desired peel and aril color. For example, Hicaznar, a pomegranate variety used in this study and also widely cultivated, has a hard-seeded cultivars although it shows superior characteristics in terms of dark red skin and other fruit characteristics. These findings show that the number of pomegranate cultivars with soft-seeded characteristics is quite low in the world and the most of them do not have other good fruit characteristics. Therefore, it is necessary to develop new pomegranate cultivars with red skin and red aryl color, and also soft-seeded features.
Development of new pomegranate varieties with desired characteristics is very difficult and takes a long period of time by conventional breeding methods. Marker assisted selection (MAS), which is among the new breeding techniques, reduce the amount of time during breeding of new fruit cultivars [43,44]. MAS allows the selection based on the genotype directly without looking at the phenotype of the plants [45]. In order to apply this technique, there is a need to develop molecular markers associated with the desired traits.
Up to date, no molecular marker to be used directly in the breeding process of pomegranates has been identified. However, there are some studies conducted for the identification of molecular markers related with some traits including soft-seed feature. For example, in a study conducted by Sarkhosh et al., [5], a correlation was tried to be found between different fruit characteristics and RAPD markers of soft-seeded pomegranate cultivars, but no correlation was found between the fruit characteristics examined and RAPD markers. In another study, molecular markers were developed associated with important horticultural traits including seed hardness by performing quantitative trait loci (QTL) mapping [46]. Although this QTL region associated with seed firmness was found to be significant in consecutive years, it has not been validated and is thought to be specific for the population studied due to genetic differences between the parents.
In recent years, especially after completion of the pomegranate genome, there have been a number of studies to find the differences between soft-and hard-seeded pomegranates at DNA, RNA, and protein level. In these studies, although some differences were determined at DNA, RNA, and protein levels between the compared genomes, no molecular marker that can be used in the breeding process of pomegranates has been determined yet [12,[21][22][23]. In this study, three RAPD markers associated with soft-and hard-seeded traits were developed. The InDel marker was also developed based on the sequences of two of these polimorfic RAPD fragments. The ability of all these markers to distinguish softseeded and hard-seeded pomegranate genotypes from each other was tested. The markers developed in this study will allow soft-seeded pomegranate genotypes to be selected in an earlier developmental stage and in a shorter time during pomegranate breeding. To our knowledge, this is the first report on the identification of molecular markers that can distinguish sof-seeded and hard-seeded pomegranate genotypes.

Conclusions
In plant breeding studies, the MAS technique is used to select individuals with desired characteristics in a short time and effectively. In this study, molecular markers associated with soft-and hard-seeded characteristics were developed for the first time to use of MAS technique in pomegranate breeding studies. Among these markers, three of them were RAPD markers and the product of one of these RAPD markers were sequenced and converted to the sequence-characterized InDel marker. All the markers developed in this study will enable pomegranate breeders to select the desired soft-seeded pomegranate genotypes easily without waiting 5-6 years for bearing fruit and will ensure that the breeding process is carried out effectively and quickly.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/life13051123/s1, Table S1: Names and sequences of RAPD primers used; Table S2: Names and sequences of SSR primers used.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article. The data are also provided in the supplementary materials, and may be shared upon request.